1. Field of the Invention
Pseudorabies (Aujeszky's disease) is caused by a herpesvirus belonging to the genus alphaherpesvirinae. It is a contagious and sometimes fatal disease of swine. Infection during gestation can result in fetal death and abortion. It is estimated that annual losses due to pseudorabies is as high as 60 million dollars in the United States. This economic impact has resulted in a decision by the swine industry and regulatory officials to eradicate the pseudorabies virus (PRV).
During the initial phase of the acute disease, PRV replicates in the upper respiratory tract. Virus can then disseminate by vascular, lymphoid and nervous tissues [D. P. Gustafson, In Diseases of Swine, ed. by A. D. Leman, et al., 6th edition, pp. 274-289, Iowa State University Press, Ames, Iowa]. Infections virus and/or viral genome can be detected from lung, tonsil, brain stem, trigeminal ganglia and peripheral blood lymphocytes [F. Wang et al., J. Leukocyte Biol. 43: 256-264 (1988); G. Wittmann et al., Arch. Virol. 66: 227-240 (1980); H. J. Rhiza In Latent Herpes Virus Infections in Veterinary Medicine, ed. by G. Wittman et al., Martinus Nijhoff publishers, The Hague pp. 429-444 (1984); H. J. Rhiza et al., In Proc. 14th International Herpes Workshop, Nyborg, Denmark, pp. 55 (1989)]. Upon cessation of clinical signs and recovery from infection, the virus is not eliminated from the animal and persists with the animal indefinitely. Sometimes, the infection is subclinical and goes unnoticed. The animal also becomes a carrier of pseudorabies. In either case, the virus exists in various cell types of the animal in a noninfectious form and is commonly known as a latent infection. The complete viral genome is present but fails to replicate fully to produce infectious virus. The latent virus can reactivate spontaneously or be induced to reactivate by exogenous stimuli, the carrier animal disseminates infectious virus to susceptible animals which may result in death of the animal or establishment of new PRV carriers. Thus, the latent virus is the source and reservoir of the disease and is regarded as an obstacle to the successful control and eradication of PRV.
This invention relates to the identification and generation of nucleic acid probes that are capable of distinguishing between productive and latent infections.
2. Description of the Prior Art
The mechanisms of establishment, maintenance and reactivation of the latent pseudorabies is not known at the present time; but it is quite clear that the latency-reactivation cycle contributes to the perpetuation of PRV in the swine population. Although several methods for detecting latent PRV infection in swine has been described, there is no available diagnostic method which is both sensitive and convenient. Direct experimental evidence for the existence of latent PRV has been obtained by in vitro reactivation of latent virus following tissue explantation [A. Sabo et al., Acta Virol. 29: 208-214 (1976); D. E. Gutekunst et al., Am. J. Vet. Res. 41: 1315-1316 (1980); G. W. Beran et al., J. Am. Vet. Med. Assoc. 176: 998-1000 (1980)]. A major drawback of this method is the need for sterile, viable tissues in the assay. This is also complicated by the use of vaccine viruses that are defective in the thymidine kinase gene, for these viruses can establish a latent infection but do not reactivate as readily. In vivo reactivation following daily administration, over a course of several days, of very high doses of corticosteroids has been used successfully to reactivate latent PRV [W. L. Mengeling, Am. J. Vet. Res. 50: 1658-1666 (1989); J. R. Van Oirschot et al., Am. J. Vet. Res. 45: 567-571 (1984); R. E. Mock et al., J. Comp. Med., 45: 56-59 (1981)]. The method does not appear to work in all cases, and the effects of the associated severe immunosuppression can be a complicating factor. Nucleic acid hybridization such as solution and blot hybridization techniques have been used [D. E. Gutekunst, Am. J. Vet. Res. 40: 1568-1572 (1972); H. J. Rhiza et al., Virology 155: 600-613 (1986); R. G. McFarlane et al., Am. J. Vet. Res. 46: 1133-1136 (1985); Belak et al., J. Vet. Med. 34: 519-529 (1987)], but they are not capable of detecting extremely low quantities of latent DNA. In situ hybridization is inherently sensitive since it can detect latency at the single cell level [H. J. Rhiza et al., supra, 1984; D. L. Rock, Arch Virol. 98: 99-106 (1988)]; unfortunately, the method is extremely labor intensive. The most promising technique to date is the polymerase chain reaction (PCR) technique capable of amplifying the target DNA sequence by a millionfold [Belak et al., Arch. Virol. 108: 279-286 (1989)]. Since the amount of PRV genome present in latently infected swine is exceedingly low, the target DNA sequence remains a limiting step for determining pseudorabies latency even with the PCR amplification method.
The PRV genome is a linear, duplex DNA molecule with a molecular weight of approximately 90.times.10.sup.6 [T. Ben-Porat et al., "Molecular Biology of Pseudorabies Virus," In B. Roizman (ed.), The Herpesviruses, Vol. 3, Plenum Publishing Corporation, New York, pp. 105-173 (1985)]. It is estimated that the genetic material is capable of coding for 50 to 100 viral genes. The transcription pattern of PRV in infected cells is extremely complex; however, the genes are expressed in a coordinated, and temporally regulated manner [L. T. Feldman et al., Virology 116: 250-263 (1982); Virology 97: 316-327 (1979); S. Ihara et al., Virology 131: 437-454 (1983); and T. Rakusanova et al., Virology 46: 877-889 (1971)]. In general, herpesvirus genes are categorized into three classes: immediate-early (IE), early, and late genes. The IE genes are transcribed immediately upon infection and do not require de novo protein synthesis. Transcription of early genes depends on IE protein expression and occurs before viral DNA replication. The late genes are transcribed after the onset of viral protein and DNA synthesis.
During herpesvirus latency, a restricted region of the viral genome is transcriptionally active. RNAs denoted as latency-associated transcripts (LATs) are detectable in animals latently infected with the virus [Stevens et al., Science 235: 1056-1059 (1987)]. For pseudorabies virus, the LATs are located downstream of the immediate-early (IE180) gene and in the antiparallel orientation. Since the pseudorabies LATs are the only genetic elements present during latency, it is expected that they play a role in the establishment, maintenance and/or reactivation of the latent virus. As such, the LATs are specific for latency and they are present in higher quantity than the latent genome; and therefore, a better target for detection.
Patent No. PCT/US86/01804 entitled "Pseudorabies Virus Deletion Mutants and Vaccines Containing Same" assigned to Syntrovet Incorporation has indicated the importance of the junction region between the unique long and internal repeat region for the attenuation of PRV. However, there is no description of its involvement in PRV latency. This is not surprising, since the latency-associated transcripts for herpes simplex virus were first described in 1987, and those for PRV were not described until 1989 [first by Cheung, J. Virol. 63: 2908-2913 (July 1989); then by Lokengard et al., Arch. Virol. 110: 129-136 (1990)].
In summary, there are no convenient diagnostic probes for PRV latency.